Skip to main content
SpringerLink
Log in

Separating the Inseparable: The Metabolomic Analysis of Plant–Pathogen Interactions

  • Protocol
  • First Online:
Plant Metabolomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 860))

Abstract

Plant–microbe interactions—whether pathogenic or symbiotic—exert major influences on plant physiology and productivity. Analysis of such interactions represents a particular challenge to metabolomic approaches due to the intimate association between the interacting partners coupled with a general commonality of metabolites. We here describe an approach based on co-cultivation of Arabidopsis cell cultures and bacterial plant pathogens to assess the metabolomes of both interacting partners, which we refer to as dual metabolomics.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. M Heil, IT Baldwin: Fitness costs of induced resistance: emerging experimental support for a slippery concept. Trends in Plant Science 2002, 7: 61–67.

    Article  PubMed  CAS  Google Scholar 

  2. L Salvaudon, T Giraud, JA Shykoff: Genetic diversity in natural populations: a fundamental component of plant-microbe interactions. Current Opinion in Plant Biology 2008, 11:135–143.

    Article  PubMed  CAS  Google Scholar 

  3. EC Oerke, H-W Dehne, F Schönbeck, A Weber: Crop production and crop protection—Estimated losses in major food and cash crops. Amsterdam: Elsevier; 1994.

    Google Scholar 

  4. M Parniske: Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews Microbiology 2008, 6:763–775.

    Article  PubMed  CAS  Google Scholar 

  5. KM Jones, H Kobayashi, BW Davies, ME Taga, GC Walker: How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nature Reviews Microbiology 2007, 5:619–633.

    Article  PubMed  CAS  Google Scholar 

  6. V Bianciotto, P Bonfante: Arbuscular mycorrhizal fungi: a specialised niche for rhizo-spheric and endocellular bacteria. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 2002, 81:365–371.

    Article  CAS  Google Scholar 

  7. RP Ryan, K Germaine, A Franks, DJ Ryan, DN Dowling: Bacterial endophytes: recent developments and applications. Fems Microbiology Letters 2008, 278:1–9.

    Article  PubMed  CAS  Google Scholar 

  8. LAJ Mur, P Kenton, AJ Lloyd, H Ougham, E Prats: The hypersensitive response; the centenary is upon us but how much do we know? Journal of Experimental Botany 2008, 59:501–520.

    Article  PubMed  CAS  Google Scholar 

  9. C Mille-Lindblom, E von Wachenfeldt, LJ Tranvik: Ergosterol as a measure of living fungal biomass: persistence in environmental samples after fungal death. Journal of Microbiological Methods 2004, 59:253–262.

    Article  PubMed  CAS  Google Scholar 

  10. D Choi, RM Bostock, S Avdiushko, DF Hildebrand: Lipid-derived signals that discriminate wound-responsive and pathogen-responsive isoprenoid pathways in plants – methyl jasmonate and the fungal elicitor arachidonic-acid induce different 3-hydroxy-3-methylglutaryl-coenzyme-a reductase genes and antimicrobial isoprenoids in Solanum-tuberosum L. Proceedings of the National Academy of Sciences of the United States of America 1994, 91:2329–2333.

    Article  PubMed  CAS  Google Scholar 

  11. K Shimizu: Metabolic flux analysis based on C-13-labeling experiments and integration of the information with gene and protein expression patterns. In: Recent Progress of Biochemical and Biomedical Engineering in Japan Ii, vol. 91. pp. 1–49. Berlin: SPRINGER-VERLAG BERLIN; 2004: 1–49.

    Google Scholar 

  12. TCR Williams, L Miguet, SK Masakapalli, NJ Kruger, LJ Sweetlove, RG Ratcliffe: Metabolic network fluxes in heterotrophic Arabidopsis cells: Stability of the flux distribution under different oxygenation conditions. Plant Physiology 2008, 148:704–718.

    Article  PubMed  CAS  Google Scholar 

  13. MK Hellerstein: New stable isotope-mass spectrometric techniques for measuring fluxes through intact metabolic pathways in mammalian systems: introduction of moving pictures into functional genomics and biochemical phenotyping. Metabolic Engineering 2004, 6:85–100.

    Article  PubMed  CAS  Google Scholar 

  14. YH Choi, HK Kim, HJM Linthorst, JG Hollander, AWM Lefeber, C Erkelens, JM Nuzillard, R Verpoorte: NMR metabolomics to revisit the tobacco mosaic virus infection in Nicotiana tabacum leaves. Journal of Natural Products 2006, 69:742–748.

    Article  PubMed  CAS  Google Scholar 

  15. J Zhao, LC Davis, R Verpoorte: Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances 2005, 23:283–333.

    Article  PubMed  CAS  Google Scholar 

  16. RA Dietrich, MH Richberg, R Schmidt, C Dean, JL Dangl: A novel zinc finger protein is encoded by the arabidopsis LSD1 gene and functions as a negative regulator of plant cell death. Cell 1997, 88:685–694.

    Article  PubMed  CAS  Google Scholar 

  17. DH Aviv, C Rusterucci, BF Holt, RA Dietrich, JE Parker, JL Dangl: Runaway cell death, but not basal disease resistance, in Isd1 is SA- and NIM1/NPR1-dependent. Plant Journal 2002, 29:381–391.

    Article  PubMed  CAS  Google Scholar 

  18. S Lorrain, F Vailleau, C Balaque, D Roby: Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants? Trends in Plant Science 2003, 8:263–271.

    Article  PubMed  CAS  Google Scholar 

  19. JD Clarke, SM Volko, H Ledford, FM Ausubel, XN Dong: Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. Plant Cell 2000, 12:2175–2190.

    Article  PubMed  CAS  Google Scholar 

  20. JR Alfano, AO Charkowski, WL Deng, JL Badel, T Petnicki-Ocwieja, K van Dijk, A Collmer: The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proceedings of the National Academy of Sciences of the United States of America 2000, 97:4856–4861.

    Article  PubMed  CAS  Google Scholar 

  21. T Kondo, S Sawa, A Kinoshita, S Mizuno, T Kakimoto, H Fukuda, Y Sakagami: A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis. Science 2006, 313:845–848.

    Article  PubMed  CAS  Google Scholar 

  22. AK Mullen, MR Clench, S Crosland, KR Sharples: Determination of agrochemical compounds in soya plants by imaging matrix-assisted laser desorption/ionisation mass spectrometry. Rapid Communications in Mass Spectrometry 2005, 19:2507–2516.

    Article  PubMed  CAS  Google Scholar 

  23. S Robinson, K Warburton, M Seymour, M Clench, J Thomas-Oates: Localization of water-soluble carbohydrates in wheat stems using imaging matrix-assisted laser desorption ionization mass spectrometry. New Phytologist 2007, 173:438–444.

    Article  PubMed  CAS  Google Scholar 

  24. JJ Jones, S Mariccor, AB Batoy, CL Wilkins: A comprehensive and comparative analysis for MALDI FTMS lipid and phospholipid profiles from biological samples. Computational Biology and Chemistry 2005, 29:294–302.

    Article  PubMed  CAS  Google Scholar 

  25. P Heraud, S Caine, G Sanson, R Gleadow, BR Wood, D McNaughton: Focal plane array infrared imaging: a new way to analyse leaf tissue. New Phytologist 2007, 173:216–225.

    Article  PubMed  Google Scholar 

  26. F Jamme, P Robert, B Bouchet, L Saulnier, P Dumas, F Guillon: Aleurone cell walls of wheat grain: High spatial resolution investigation using synchrotron infrared microspectroscopy. Applied Spectroscopy 2008, 62:895–900.

    Article  PubMed  CAS  Google Scholar 

  27. BO Budevska, ST Sum, TJ Jones: Application of Multivariate curve resolution for analysis of FT-IR microspectroscopic images of in situ plant tissue. Applied Spectroscopy 2003, 57:124–131.

    Article  PubMed  CAS  Google Scholar 

  28. Z Movasaghi, S Rehman, IU Rehman: Raman Spectroscopy of Biological Tissues. Applied Spectroscopy Reviews 2007, 42:493–541.

    Article  CAS  Google Scholar 

  29. N Gierlinger, M Schwanninger: Chemical imaging of poplar wood cell walls by confocal Raman microscopy. Plant Physiology 2006, 140:1246–1254.

    Article  PubMed  CAS  Google Scholar 

  30. AJ Lloyd, JW Allwood, CL Winder, WB Dunn, JK Heald, SM Cristescu, A Sivakumaran, FJM Harren, J Mulema, K Denby, R Goodacre, AR Smith, LAJ Mur: Metabolomic approaches reveal that cell wall modifications play a major role in ethylene-mediated resistance against Botrytis cinerea. Plant Journal 2011, 67:852–868.

    Google Scholar 

  31. PF McCabe, CJ Leaver: Programmed cell death in cell cultures. Plant Molecular Biology 2000, 44:359–368.

    Article  PubMed  CAS  Google Scholar 

  32. NM Cecchini, MI Monteoliva, F Blanco, L Holuigue, ME Alvarez: Features of basal and race-specific defences in photosynthetic Arabidopsis thaliana suspension cultured cells. Molecular Plant Pathology 2009, 10:305–310.

    Article  PubMed  CAS  Google Scholar 

  33. A Clarke, R Desikan, RD Hurst, JT Hancock, SJ Neill: NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures. Plant Journal 2000, 24:667–677.

    Article  PubMed  CAS  Google Scholar 

  34. P Cossart, PJ Sansonetti: Bacterial invasion: The paradigms of enteroinvasive pathogens. Science 2004, 304:242–248.

    Article  PubMed  CAS  Google Scholar 

  35. MJ May, CJ Leaver: Oxidative stimulation of glutathione synthesis in Arabidopsis-thaliana Suspension-Cultures. Plant Physiology 1993, 103:621–627.

    PubMed  CAS  Google Scholar 

  36. MJ Zhou, ZJ Diwu, N PanchukVoloshina, RP Haugland: A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Analytical Biochemistry 1997, 253:162–168.

    Article  PubMed  CAS  Google Scholar 

  37. M Delledonne, YJ Xia, RA Dixon, C Lamb: Nitric oxide functions as a signal in plant disease resistance. Nature 1998, 394:585–588.

    Article  PubMed  CAS  Google Scholar 

  38. IR Davies, XJ Zhang: Nitric oxide selective electrodes. Globins and Other Nitric Oxide-Reactive Proteins, Pt A 2008, 436:63–95.

    Article  CAS  Google Scholar 

  39. E Prats, LAJ Mur, R Sanderson, TLW Carver: Nitric oxide contributes both to papilla-based resistance and the hypersensitive response in barley attacked by Blumeria graminis f. sp hordei. Molecular Plant Pathology 2005, 6:65–78.

    Google Scholar 

  40. Sambrook J., Russell D.: Molecular cloning : A laboratory manual: Cold Spring Harbor Laboratory Press; 2006.

    Google Scholar 

  41. JW Allwood, DI Ellis, R Goodacre: Biomarker metabolites capturing the metabolite variance present in a rice plant developmental period. Physiologia Plantarum 2008, 132:117–135.

    PubMed  CAS  Google Scholar 

  42. RD Hall: Plant metabolomics: from holistic hope, to hype, to hot topic. New Phytologist 2006, 169:453–468.

    Article  PubMed  CAS  Google Scholar 

  43. O Fiehn, J Kopka, P Dormann, T Altmann, RN Trethewey, L Willmitzer: Metabolite profiling for plant functional genomics. Nature Biotechnology 2000, 18:1157–1161.

    Article  PubMed  CAS  Google Scholar 

  44. S Vaidyanathan, DB Kell, R Goodacre: Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification. J Am Soc Mass Spectrom 2002, 13:118–128.

    Article  Google Scholar 

  45. JW Allwood, DI Ellis, JK Heald, R Goodacre, LAJ Mur: Metabolomic approaches reveal that phosphatidic and phosphatidyl glycerol phospholipids are major discriminatory non-polar metabolites in responses by Brachypodium distachyon to challenge by Magnaporthe grisea. Plant Journal 2006, 46:351–368.

    Article  PubMed  CAS  Google Scholar 

  46. V Houot, P Etienne, A-S Petitot, S Barbier, J-P Blein, L Suty: Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner J. Exp. Bot. 2001, 52:1721–1730.

    Article  CAS  Google Scholar 

  47. E Kombrink, K Hahlbrock: Responses of cultured parsley cells to elicitors from phytopathogenic fungi : Timing and dose dependency of elicitor-induced reactions Plant Physiol. 1986, 81:216–221.

    Article  PubMed  CAS  Google Scholar 

  48. A Levine, R Tenhaken, R Dixon, C Lamb: H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 1994, 79:583–593.

    Article  PubMed  CAS  Google Scholar 

  49. C Lodewyckx, J Vangronsveld, F Porteous, ERB Moore, S Taghavi, M Mezgeay, D van der Lelie: Endophytic bacteria and their potential applications. Critical Reviews in Plant Sciences 2002, 21:583–606.

    Article  Google Scholar 

  50. R Goodacre, EM Timmins, R Burton, N Kaderbhai, AM Woodward, DB Kell, PJ Rooney: Rapid identification of urinary tract infection bacteria using hyperspectral whole-organism fingerprinting and artificial neural networks. Microbiology-Sgm 1998, 144:1157–1170.

    Article  CAS  Google Scholar 

  51. NN Kaderbhai, DI Broadhurst, DI Ellis, R Goodacre, DB Kell: Functional genomics via metabolic footprinting: monitoring metabolite secretion by Escherichia coli tryptophan metabolism mutants using FT-IR and direct injection electrospray mass spectrometry. Comparative and Functional Genomics 2003, 4:376–391.

    Article  PubMed  CAS  Google Scholar 

  52. FM Carrau, K Medina, L Farina, E Boido, PA Henschke, E Dellacassa: Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains. Fems Yeast Research 2008, 8:1196–1207.

    Article  PubMed  CAS  Google Scholar 

  53. R Dowlatabadia, AM Weljie, TA Thorpe, EC Yeung, HJ Vogel: Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy. Plant Physiology and Biochemistry 2009, 47:343–350.

    Article  Google Scholar 

  54. CL Winder, WB Dunn, S Schuler, D Broadhurst, R Jarvis, GM Stephens, R Goodacre: Global metabolic profiling of Escherichia coli cultures: An evaluation of methods for quenching and extraction of intracellular metabolites. Analytical Chemistry 2008, 80:2939–2948.

    Article  PubMed  CAS  Google Scholar 

  55. J Fliegmann, A Mithofer, G Wanner, J Ebel: An ancient enzyme domain hidden in the putative beta-glucan elicitor receptor of soybean may play an active part in the perception of pathogen-associated molecular patterns during broad host resistance. Journal of Biological Chemistry 2004, 279:1132–1140.

    Article  PubMed  CAS  Google Scholar 

  56. D Nennstiel, D Scheel, T Nurnberger: Characterization and partial purification of an oligopeptide elicitor receptor from parsley (Petroselinum crispum). Febs Letters 1998, 431:405–410.

    Article  PubMed  CAS  Google Scholar 

  57. L Gomez-Gomez, T Boller: Flagellin perception: a paradigm for innate immunity. Trends in Plant Science 2002, 7:PII S1360-1385(02)02261-6.

    Google Scholar 

  58. A Meyer, A Puhler, K Niehaus: The lipopolysaccharides of the phytopathogen Xanthomonas campestris pv. campestris induce an oxidative burst reaction in cell cultures of Nicotiana tabacum. Planta 2001, 213:214–222.

    Google Scholar 

  59. J Lee, DF Klessig, T Nurnberger: A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity. Plant Cell 2001, 13:1079–1093.

    Article  PubMed  CAS  Google Scholar 

  60. S Bourque, MN Binet, M Ponchet, A Pugin, A Lebrun-Garcia: Characterization of the cryptogein binding sites on plant plasma membranes. Journal of Biological Chemistry 1999, 274:34699–34705.

    Article  PubMed  CAS  Google Scholar 

  61. DA Navarre, TJ Wolpert: Victorin induction of an apoptotic/senescence-like response in oats. Plant Cell 1999, 11:237–249.

    Article  PubMed  CAS  Google Scholar 

  62. M Rohe, A Gierlich, H Hermann, M Hahn, B Schmidt, S Rosahl, W Knogge: The race-specific elicitor, Nip1, from the barley pathogen, Rhynchosporium-Secalis, determines avirulence on host plants of the Rrs1 resistance genotype. Embo Journal 1995, 14:4168–4177.

    PubMed  CAS  Google Scholar 

  63. W D’Haeze, M Holsters: Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology 2002, 12:79R–105R.

    Article  PubMed  Google Scholar 

  64. M Parniske, JA Downie: Plant biology – Locks, keys and symbioses. Nature 2003, 425:569–570.

    Article  PubMed  CAS  Google Scholar 

  65. M de Torres, JW Mansfield, N Grabov, IR Brown, H Ammouneh, G Tsiamis, A Forsyth, S Robatzek, M Grant, J Boch: Pseudomonas syringae effector AvrPtoB suppresses basal defence in Arabidopsis. Plant Journal 2006, 47:368–382.

    Article  Google Scholar 

  66. RZ Li, XY Jia, X Mao: Ethanol-inducible gene expression system and its applications in plant functional genomics. Plant Science 2005, 169:463–469.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank the UK BBSRC for partly funding this work through studentships to JWA and AJL. JWA and RG are also indebted to the EU Framework VI funded project META-PHOR (FOOD-CT-2006-036220).

Author information

Authors and Affiliations

  1. IBERS – Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, UK

    J. William Allwood, Jim Heald, Amanda J. Lloyd & Luis A. J. Mur

  2. School of Chemistry, Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, UK

    J. William Allwood & Royston Goodacre

  3. Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, UK

    Royston Goodacre

Authors
  1. J. William Allwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jim Heald
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amanda J. Lloyd
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Royston Goodacre
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luis A. J. Mur
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luis A. J. Mur .

Editor information

Editors and Affiliations

  1. , Dept. Computer Sciences, Aberystwyth University, King Street, Aberystwyth, SY23 3DB, United Kingdom

    Nigel W. Hardy

  2. Plant Research International, Wageningen, Netherlands

    Robert D. Hall

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Allwood, J.W., Heald, J., Lloyd, A.J., Goodacre, R., Mur, L.A.J. (2011). Separating the Inseparable: The Metabolomic Analysis of Plant–Pathogen Interactions. In: Hardy, N., Hall, R. (eds) Plant Metabolomics. Methods in Molecular Biology, vol 860. Humana Press. https://doi.org/10.1007/978-1-61779-594-7_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-594-7_3

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-593-0

  • Online ISBN: 978-1-61779-594-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation